Physicochemical stimuli to enhance the quality of human engineered cartilage: the role of osmolarity and calcium

Due to the low regenerative capacity of articular cartilage, regenerative approaches are needed to treat cartilage defects and to restore the function of the tissue in the joint. However, a general drawback of current cartilage replacement tissues is an insufficient deposition of its main molecular components, type II collagen and proteoglycan. As a result, the tissue cannot withstand the demanding mechanical conditions in the joint. Recent studies of our group achieved an acute stimulation of cartilage matrix synthesis by a defined mechanical loading protocol which depended on the tissue’s glycosaminoglycan (GAG)-content and its associated fixed charge density (FCD). However, to what extend mechano-induced physicochemical sub-stimuli contribute to cartilage matrix production remains unclear. Identifying the decisive sub-parameter that contributes to load-induced stimulation of cartilage matrix synthesis would provide an easily applicable stimulus to optimize the quality of cartilage replacement tissues. Due to the essential role of osmotic pressure for cartilage function, hyperosmotic challenge appears as an important sub-parameter of the loading-response. However, the contribution of acute hyperosmotic pressure to cartilage homeostasis is unclear and models that take a cartilage typical FCD into consideration are required. Interestingly, long-term maturation of animal chondrocytes under hyperosmotic conditions enhanced the matrix content of engineered cartilage but this was so far never investigated for human 3D-cultured chondrocytes. Thus, the aim of this this study was to elucidate whether acute hyperosmotic stimulation, as a sub-parameter of mechanical compression, regulates cartilage matrix synthesis in a human engineered cartilage model at low and high FCD. In parallel, it was investigated whether long-term hyperosmotic stimulation can enhance the matrix synthesis and deposition of cartilage replacement tissue. To achieve these aims, human engineered cartilage was pre-matured for 3 or 35 days to develop a cartilage-like matrix of low or high FCD. Acute hyperosmotic stimulation on day 3 and on day 35 for 3 to 24 hours indicated that the known mechano-response markers ERK1/2, p38, NFAT5, FOS and FOSB are also immediate osmo-response markers, irrespective of the FCD content of the tissue. Opposite to previous results from mechanical loading studies, a downregulation of pro-chondrogenic SOX9 protein and BMP pathway activity indicated an anti-chondrogenic effect of short-term hyperosmotic stimulation on chondrocytes. However, this did not lead to changes in cartilage matrix synthesis at low and at high FCD. Thus, although acute hyperosmotic stimulation and mechanical compression partly triggered similar response pathways, short-term hyperosmotic pressure was no major player to influence the regulation of cartilage matrix synthesis. In the context of long-term hyperosmotic stimulation, previous studies suggested a role of the extracellular calcium microenvironment for cartilage matrix synthesis and deposition. Since articular chondrocytes (AC) and mesenchymal stromal cells (MSC) are often used cell types for the design of cartilage replacement tissues, the response of both cell types to long-term hyperosmotic stimulation was investigated using extracellular calcium. Interestingly, the here obtained data revealed that long-term hyperosmotic calcium stimulation for 35 days compromised cartilage matrix formation in AC-based cartilage replacement tissue but promoted the cartilage matrix formation in neocartilage generated from MSC. Investigation of pro- and anti-chondrogenic signaling pathways after long-term calcium stimulation indicated a specific induction of catabolic S100A4 and PTHLH expression in AC. Stimulating AC with recombinant human PTHrP1-34 peptide partly reproduced the calcium-mediated reduction of cartilage matrix deposition, suggesting a role of PTHrP for impaired cartilage matrix formation. Importantly, the inverse regulation of GAG synthesis in AC and MSC-derived chondrocytes was calcium-specific and not caused by general hyperosmotic effects. Long-term extracellular calcium stimulation, therefore, provides a novel means to enhance the cartilage matrix content of MSC-based engineered cartilage whereas such conditions should be avoided during AC neocartilage formation. Overall, this study provides important information on the role of physicochemical stimuli for cartilage matrix formation in human engineered cartilage. It was demonstrated that acute hyperosmotic pressure was no effective stimulus to influence cartilage matrix synthesis, and further studies are now needed to determine the contribution other load-induced sub-parameter for GAG synthesis. Furthermore, this study indicated that long-term high extracellular calcium treatment provides a novel means to enhance the quality of MSC-based cartilage replacement tissue by stimulating GAG synthesis and GAG deposition. For the application in osteochondral tissue engineering approaches, this implies that MSC should be the first choice for cartilage matrix deposition in the vicinity of resorbable calcified bone replacement materials. However, studies are now needed to confirm the here observed effects of soluble extracellular calcium using resorbable bone replacement material as a potential calcium source

Human Autoantibody Silencing by Immunoglobulin Light Chains

Several newly arising human antibodies are polyreactive, but in normal individuals the majority of these potentially autodestructive antibodies are removed from the repertoire by receptor editing or B cell deletion in the bone marrow. To determine what proportion of naturally arising autoantibodies can be silenced by immunoglobulin (Ig) light chain receptor editing, we replaced the light chains in 12 such antibodies with a panel of representative Igκ and Igλ chains. We found that most naturally arising autoantibodies are readily silenced by light chain exchange. Thus, receptor editing may account for most autoreactive antibody silencing in humans. Light chain complementarity determining region (CDR) isoelectric points did not correlate with silencing activity, but Igλ genes were more effective than Igκ genes as silencers. The greater efficacy of Igλ chains as silencer of autoreactivity provides a possible explanation for the expansion and altered configuration of the Igλ locus in evolution

Defective B cell tolerance checkpoints in systemic lupus erythematosus

A cardinal feature of systemic lupus erythematosus (SLE) is the development of autoantibodies. The first autoantibodies described in patients with SLE were those specific for nuclei and DNA, but subsequent work has shown that individuals with this disease produce a panoply of different autoantibodies. Thus, one of the constant features of SLE is a profound breakdown in tolerance in the antibody system. The appearance of self-reactive antibodies in SLE precedes clinical disease, but where in the B cell pathway tolerance is first broken has not been defined. In healthy humans, autoantibodies are removed from the B cell repertoire in two discrete early checkpoints in B cell development. We found these checkpoints to be defective in three adolescent patients with SLE. 25–50% of the mature naive B cells in SLE patients produce self-reactive antibodies even before they participate in immune responses as compared with 5–20% in controls. We conclude that SLE is associated with abnormal early B cell tolerance

Effects of communication training with the MAAS-Global-D instrument on the antibiotic prescribing for respiratory infections in primary care: study protocol of a randomised controlled trial

BACKGROUND: Primary care physicians account for the majority of antibiotic prescribing in ambulatory care in Germany. Respiratory diseases are, regardless of effectiveness, often treated with antibiotics. Research has found this use without indication to be caused largely by communication problems (e.g. expectations on the patient’s part or false assumptions about them by the physician). The present randomised controlled trial (RCT) study evaluates whether communication training for primary care physicians can reduce the antibiotic prescribing rate for respiratory tract infections. METHODS/DESIGN: The study consists of three groups: group A will receive communication training; group B will be given the same, plus additional, access to an evidence-based point-of-care tool; and group C will function as the control group. The primary endpoint is the difference between intervention and control groups regarding the antibiotic prescribing rate before and after the intervention assessed through routine data. The communication skills are captured with the help of the communication instrument MAAS-Global-D, as well as individual videos of physician-patient consultations recorded by the primary care physicians. These skills will also be regarded with respect to the antibiotic prescribing rate. A process evaluation using qualitative as well as quantitative methods should provide information about barriers and enablers to implementing the communication training. DISCUSSION: The trial contributes to an insight into the effectiveness of the different components to reduce antibiotic prescribing, which will also be supported by an extensive evaluation. Communication training could be an effective method of reducing antibiotic prescribing in primary care. TRIAL REGISTRATION: DRKS00009566 DATE REGISTRATION: 5 November 2015

Impact of treatment intensity on infectious complications in patients with acute myeloid leukemia

Abstract Background Infectious complications reflect a major challenge in the treatment of patients with acute myeloid leukemia (AML). Both induction chemotherapy and epigenetic treatment with hypomethylating agents (HMA) are associated with severe infections, while neutropenia represents a common risk factor. Here, 220 consecutive and newly diagnosed AML patients were analyzed with respect to infectious complications dependent on treatment intensity and antifungal prophylaxis applied to these patients. Patients and methods We retrospectively analyzed 220 patients with newly diagnosed AML at a tertiary care hospital between August 2016 and December 2020. The median age of AML patients undergoing induction chemotherapy ( n  = 102) was 61 years (25–76 years). Patients receiving palliative AML treatment ( n  = 118) had a median age of 75 years (53–91 years). We assessed the occurrence of infectious complication including the classification of pulmonary invasive fungal disease (IFD) according to the EORTC/MSG criteria at diagnosis and until day 100 after initiation of AML treatment. Furthermore, admission to intensive care unit (ICU) and subsequent outcome was analyzed for both groups of AML patients, respectively. Results AML patients subsequently allocated to palliative AML treatment have a significantly higher risk of pneumonia at diagnosis compared to patients undergoing induction chemotherapy (37.3% vs. 13.7%, P   2, P  < 0.001, respectively) in this intent-to-treat analysis. Conclusion The risk and the pattern of infectious complications at diagnosis and after initiation of AML therapy depends on age, ECOG performance status and subsequent treatment intensity. A comprehensive diagnostic work-up for identification of pulmonary IFD is indispensable for effective treatment of pneumonia in AML patients. The presence of infectious complications at diagnosis contributes to an inferior outcome in elderly AML patients

Clinical experience with venetoclax in patients with newly diagnosed, relapsed, or refractory acute myeloid leukemia

Background Diagnosis of acute myeloid leukemia (AML) is associated with poor outcome in elderly and unfit patients. Recently, approval of the BCL-2 inhibitor venetoclax (VEN) in combination with hypo-methylating agents (HMA) led to a significant improvement of response rates and survival. Further, application in the relapsed or refractory (r/r) AML setting or in context of allogeneic stem cell transplantation (alloHSCT) seems feasible. Methods and patients Fifty-six consecutive adult AML patients on VEN from January 2019 to June 2021 were analyzed retrospectively. Patients received VEN either as first-line treatment, as subsequent therapy (r/r AML excluding prior alloHSCT), or at relapse after alloHSCT. VEN was administered orally in 28-day cycles either combined with HMA or low-dose cytarabine (LDAC). Results After a median follow-up of 11.5 (range 6.1–22.3) months, median overall survival (OS) from start of VEN treatment was 13.3 (2.2–20.5) months, 5.0 (0.8–24.3) months and 4.0 (1.5–22.1) months for first-line, subsequent line treatment and at relapse post-alloHSCT, respectively. Median OS was 11.5 (10–22.3) months from start of VEN when subsequent alloHSCT was carried out. Relapse-free survival (RFS) for the total cohort was 10.2 (2.2 – 24.3) months. Overall response rate (composite complete remission + partial remission) was 51.8% for the total cohort (61.1% for VEN first-line treatment, 52.2% for subsequent line and 42.8% at relapse post-alloHSCT). Subgroup analysis revealed a significantly reduced median OS in FLT3-ITD mutated AML with 3.4 (1.9–4.9) months versus 10.4 (0.8–24.3) months for non-mutated cases, (HR 4.45, 95% CI 0.89–22.13, p  = 0.0002). Patients harboring NPM1 or IDH1/2 mutations lacking co-occurrence of FLT3-ITD showed a survival advantage over patients without those mutations (11.2 (5–24.3) months versus 5.0 (0.8–22.1) months, respectively, (HR 0.53, 95% CI 0.23 – 1.21, p  = 0.131). Multivariate analysis revealed mutated NPM1 as a significant prognostic variable for achieving complete remission (CR) (HR 19.14, 95% CI 2.30 – 436.2, p  < 0.05). The most common adverse events were hematological, with grade 3 and 4 neutropenia and thrombocytopenia reported in 44.6% and 14.5% of patients, respectively. Conclusion Detailed analyses on efficacy for common clinical scenarios, such as first-line treatment, subsequent therapy (r/r AML), and application prior to and post-alloHSCT, are presented. The findings suggest VEN treatment combinations efficacious not only in first-line setting but also in r/r AML. Furthermore, VEN might play a role in a subgroup of patients with failure to conventional chemotherapy as a salvage regimen aiming for potential curative alloHSCT